CN102827895A - Double-liquid-phase fermentation method of coupling in-situ extraction antrodia camphorate active product antrondin C - Google Patents
Double-liquid-phase fermentation method of coupling in-situ extraction antrodia camphorate active product antrondin C Download PDFInfo
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Abstract
The invention discloses a double-liquid-phase fermentation method of coupling in-situ extraction antrodia camphorate active product antrondin C. According to the method, different surfactants are added into a liquid culture medium for changing the permeability of an antrodia camphorate bacterial cell membrane, the negative feedback inhibition effect of the target product antrondin C synthesis is reduced, on the basis, different extracting agents are coupled for carrying out in-situ extraction double-liquid-phase fermentation, and active products which are secreted from cells and attached on the bacterial ball surface are extracted into the extracting agents, so the negative feedback inhibition intensity of the target product is reduced, the surfactant coupling in-situ extraction fermentation technology is adopted, the efficient synthesis and extraction of the antrodia camphorate active product can be realized in the same fermentation period, the yield of the antrodia camphorate active product antrondin C can be obviously improved, the active product in the extraction agents is extracted into ethanol phases through ethanol extraction agents, the post-processing process is simple, the extraction and the purification of the active product are convenient, and the production cost can be effectively reduced.
Description
Technical field
The present invention relates to the fermentation process of a kind of camphor tree sesame active result Antrondin C, the liquid state fermentation that particularly will regulate the cell permeability combines with the original position extraction phase, sets up the suitable biliquid extractive fermentation system that is coupled, and belongs to field of fermentation engineering.
Technical background
Camphor tree sesame (Antrodia camphorata) is name Cinnamomum kanahirai hay mushroom, red camphor tree sesame also, is the extreme precious edible medicinal fungus mushroom class of a kind of Chinese medicine circle, and its sporophore only parasitizes the hollow core timber inwall of Taiwan endemic tree Cinnamomum kanahirai hay tree, has strong camphor tree fragrance.
Between the several years in the past; Because the camphor tree sesame is pharmacologically active and hypotoxicity speciality widely; It is received publicity internationally gradually; Also paid attention to by scholar of each side and industry and extract the natural goods with drug effect by it, existing numerous now research units drop into manpower and material resources, are devoted to the research of camphor tree sesame bacterium activeconstituents.Camphor tree sesame artificial culture roughly can be divided into three kinds of cultivation basswood methods, plate method, solution fermentation; Wherein sporophore of " cultivation basswood method " acquisition and wild camphor tree sesame composition are formed basically identical; But cultivation basswood method growth cycle is long, yields poorly, and is difficult to remedy the great demand in market; If camphor tree sesame bacterial classification is carried out solid-state cultivation with the space bag, space includes fibre object, carbohydrate, dairy products etc., and it is higher to cultivate cost; And successfully realized camphor tree sesame thalline artificial culture fast through the liquid state fermentation of camphor tree sesame cell deep layer at present, so liquid state fermentation production camphor tree sesame active result will be a kind of method effectively.
Taiwan, Japanese side document have report; The physiological active products that the liquid state fermentation of camphor tree sesame obtains is except common material such as polysaccharide, triterpenes; People such as Nakamura also isolate 5 kinds of physiological active products toxilic acids and succinic acid derivative (Antrondin A-E) in the mycelium of camphor tree sesame liquid state fermentation; Wherein Antrondin B and Antrondin C have the drug effect that suppresses lung cancer LLC cell; And in the research of other drug effects, find that physiological active products Antrondin A-E also has goodish curative effect for anti-liver inflammation aspect, so such physiological active products has a extensive future.But both at home and abroad about liquid fermentation method production and to improve the research of these physiological active products output considerably less, particularly aspect the fermentation control of camphor tree sesame liquid state fermentation composite reactive product A ntrondins.
Publication number is that CN 102321684 A patent documentations such as (the biosynthetic fermentation process of a kind of promotion camphor tree sesame active result Antrondin C) discloses the output of camphor tree sesame solid state fermentation through metabolic regulation raising Antrondins compounds at present; Or the liquid state fermentation of camphor tree sesame improves contents such as triterpenes or polysaccharose substance through metabolic regulation; Not about the patent documentation report of camphor tree sesame liquid state fermentation composite reactive product A ntrondins compounds, do not improve the process patent report of active result Antrondins compounds output through regulation and control liquid state fermentation yet.
In conventional liquid fermentation method, camphor tree sesame liquid state fermentation composite reactive product A ntrondins compounds output is very low, is difficult to realize suitability for industrialized production, has become this hot research fields so how to improve the output of camphor tree sesame active result effectively.
Summary of the invention
The present invention is directed to the conventional liquid state fermentation synthetic of camphor tree sesame physiological active products yields poorly and problems such as kind is few; A kind of biliquid that adopts tensio-active agent coupling original position extractive fermentation to produce camphor tree sesame active result fermentation process that is coupled is provided; Utilize tensio-active agent to change the permeability of cytolemma earlier; Reduce the negative feedback inhibition effect of camphor tree sesame physiological active products, can improve the kind and the output of camphor tree sesame physiological active products effectively, and during the fermentation because this type of product is insoluble in water; So from emiocytosis come out still attached to the active result on bacterium ball surface can through the extraction agent extracting and enriching to extraction agent mutually; Thereby in same fermentation period, realize the efficient synthetic and extraction of camphor tree sesame active result, extraction agent mutually in active result through alcohol extraction to ethanol mutually in, make that the extraction purifying of camphor tree sesame physiological active products is easy; The extraction agent that reclaims can reuse after removing residual ethanol through distillation, thereby has reduced production cost.
For reaching goal of the invention, the present invention adopts following technical scheme:
Camphor tree sesame (Antrodia camphorata) bacterial classification is through the liquid seed culture medium enlarged culturing; Be connected in the liquid fermentation medium with certain inoculum size; At rotating speed is 80～180 rev/mins, under 22～34 ℃ of conditions of temperature, and fermentation culture 8～12 days; Adopt during the fermentation and add different sorts tensio-active agent and extraction agent; Set up the biliquid phase fermentation system of regulating the extraction of cell permeability coupling original position, reduce the intensity of physiological active products negative feedback inhibition, improve camphor tree sesame active result output.Wherein used liquid fermentation medium is: glucose 50～100g/L, analysis for soybean powder 5～15g/L, corn starch 2～6g/L, sal epsom 0.1～1.5g/L, potassium hydrogenphosphate 0.1～1.5g/L.
Used tensio-active agent is that Span series, Tween are serial, Triton, PEG series, Chinese honey locust etc., can change the permeability of camphor tree sesame cytolemma, wherein preferred Span-80.
According to optimized technical scheme of the present invention, the interpolation time of tensio-active agent Span-80 is that the liquid state fermentation of camphor tree sesame began the back 24～96 hours, and its adding proportion is 0.1%～15%.
The extraction agent kind of selecting for use has long-chain unsaturated fatty acidss such as oleic acid and nonane, octane, the isometric paraffinic of dodecyl; This kind of extractants itself is less to thalline toxicity; And be insoluble in water, can with attached to camphor tree sesame bacterium ball surfactivity product extracting and enriching to extraction agent mutually in, with the extracellular products that reduces high density to its excretory restraining effect; Increase substantially the efficient of camphor tree sesame liquid state fermentation composite reactive product, its preferred dodecyl.
According to optimized technical scheme of the present invention, the interpolation time of extraction agent dodecyl is to add during the fermentation behind the tensio-active agent 12～48 hours, and its addition is 5%～30%.
The present invention has realized that adjusting permeability of cell membrane and original position extractive fermentation are coupled; Add tensio-active agent Span-80 during the fermentation, this material has certain restraining effect to camphor tree sesame thalli growth, but it has improved the permeability of camphor tree sesame cytolemma preferably; Make most of active result in born of the same parents, transfer to outside the born of the same parents; Reduce active result negative feedback inhibition effect in the born of the same parents, on the basis of adding tensio-active agent Span-80, add the extraction agent dodecyl, going under the emulsifying effect of tensio-active agent; Dodecyl can contact with the thalline thorough mixing in the fermented liquid; Can with the continuous extracting and enriching of active result of camphor tree sesame bacterium ball surface attachment to dodecyl mutually in, thereby in same fermentation period, realize the efficient synthetic and extraction of camphor tree sesame active result, increase substantially the output of camphor tree sesame active result.
The present invention adopts tensio-active agent coupling original position extractive fermentation technology, the active result extraction that the liquid state fermentation of camphor tree sesame is generated get into the extraction agent dodecyl mutually in, be up to 400～450mg/L; Improved 300～350% than blank group, improved the output of camphor tree sesame active result greatly, the active result of dodecyl in mutually through alcohol extraction to ethanol mutually in; Make that the extraction purifying of camphor tree sesame active result is easy; The dodecyl that reclaims can reuse after removing residual ethanol through distillation, thereby has reduced production cost, and this process is simple; Can realize the heavy industrialization application, have excellent industrial application foreground.
Embodiment
Following examples bacterial strain uses therefor and cell cultures are said according to such scheme, adopt and regulate cell permeability coupling original position extractive fermentation production camphor tree sesame active result, further specify embodiments of the invention below.
Embodiment one:
The culture medium prescription of present embodiment is:
Slant medium: yam 200g/L, glucose 20g/L, sal epsom 0.1g/L, potassium primary phosphate 0.5g/L, peptone 5g/L, agar 20g/L.
Liquid seed culture medium: glucose 20g/L, analysis for soybean powder 4g/L, corn starch 2g/L, sal epsom 0.1g/L, potassium primary phosphate 0.5g/L.
Liquid fermentation medium: glucose 30g/L, analysis for soybean powder 5g/L, corn starch 4g/L, sal epsom 0.1g/L, potassium primary phosphate 0.5g/L.
Camphor tree sesame bacterial classification is transferred in the slant medium cultivates, 30 ℃ of culture temperature cultivated for 1 week; Get fresh camphor tree sesame bacterium, aseptic technique washes spore with a certain amount of sterilized water from the inclined-plane; The preparation spore suspension is equipped with in the 500ml triangular flask of 100ml liquid seed culture medium with the inoculum size access of volume(tric)fraction 10%, places 30 ℃ of constant temperature shaking tables; Rotating speed is 110 rev/mins, cultivates 3 days, obtains being in the camphor tree sesame seed liquor of growth logarithmic phase; Be that 15% inoculum size inserts and is equipped with in the 500ml triangular flask of 100ml liquid state fermentation substratum with seed liquor with volume(tric)fraction again, place on 30 ℃ of constant temperature shaking tables concussion to cultivate, rotating speed is 110 rev/mins; After the cell cultures 48 hours, in nutrient solution, add a certain amount of different sorts tensio-active agent, continue to cultivate and finished in 8 days to ferment; The different sorts tensio-active agent has different influences to camphor tree sesame liquid state fermentation composite reactive product A ntrondin C; Wherein show activity agent Span-80 effect is remarkable, adds back camphor tree sesame active result Antrondin C output and reaches 220mg/L, compares than the blank group that does not add tensio-active agent and has improved 80%.
Embodiment two:
Identical with the step of the foregoing description one; Different is: cell cultures is in the time of 48 hours; The adding volume(tric)fraction is 0.1% tensio-active agent Span-80 in nutrient solution; Continue to cultivate and finished fermentation in 8 days, active result Antrondin C output is 200mg/L, compares with the blank group and has improved 50%.
Embodiment three:
Identical with the step of the foregoing description one; Different is: cell cultures is in the time of 48 hours; The adding volume(tric)fraction is 0.3% tensio-active agent Span-80 in nutrient solution; Continue to cultivate and finished fermentation in 8 days, active result Antrondin C output is 250mg/L, compares with the blank group and has improved 90%.
Embodiment four:
Identical with the step of the foregoing description one; Different is: cell cultures is in the time of 48 hours; The adding volume(tric)fraction is 0.5% tensio-active agent Span-80 in nutrient solution; Continue to cultivate and finished fermentation in 8 days, active result Antrondin C output is 300mg/L, compares with the blank group and has improved 130%.
Embodiment five:
Identical with the step of the foregoing description one; Different is: cell cultures is in the time of 48 hours; The adding volume(tric)fraction is 0.7% tensio-active agent Span-80 in nutrient solution; Continue to cultivate and finished fermentation in 8 days, active result Antrondin C output is 150mg/L, compares with the blank group to improve and knows clearly 15%.
Embodiment six:
Identical with the step of the foregoing description four, different is: in the time of 48 hours, in nutrient solution, adding volume(tric)fraction is 0.5% tensio-active agent Span-80 in cell cultures; When cultivating 24 hours again; Add a certain amount of different sorts extraction agent, continue to cultivate again and finished fermentation in 6 days, wherein the dodecyl effect of extracting is best; The output of active result Antrondin C is 350mg/L, compares with the blank group and has improved 190%.
Embodiment seven:
Identical with the step of the foregoing description four; Different is: in the time of 48 hours, in nutrient solution, adding volume(tric)fraction is 0.5% tensio-active agent Span-80, when cultivating 24 hours again in cell cultures; The interpolation volume(tric)fraction is 5% extraction agent dodecyl; Continue training again and finished fermentation in 6 days, the output of active result Antrondin C is 250mg/L, compares with the blank group and has improved 105%.
Embodiment eight:
Identical with the step of the foregoing description four; Different is: in the time of 48 hours, in nutrient solution, adding volume(tric)fraction is 0.5% tensio-active agent Span-80, when cultivating 24 hours again in cell cultures; The interpolation volume(tric)fraction is 10% extraction agent dodecyl; Continue to cultivate again and finished fermentation in 6 days, the output of active result Antrondin C is 400mg/L, compares with the blank group and has improved 230%.
Embodiment nine:
Identical with the step of the foregoing description four; Different is: in the time of 48 hours, in nutrient solution, adding volume(tric)fraction is 0.5% tensio-active agent Span-80, when cultivating 24 hours in cell cultures; The interpolation volume(tric)fraction is 15% extraction agent dodecyl; Continue to cultivate again and finished fermentation in 6 days, the output of active result Antrondin C is 470mg/L, compares with the blank group and has improved 290%.
Embodiment ten:
Identical with the step of the foregoing description four; Different is: in the time of 48 hours, in nutrient solution, adding volume(tric)fraction is 0.5% tensio-active agent Span-80, when cultivating 24 hours in cell cultures; The interpolation volume(tric)fraction is 20% extraction agent; Continue to cultivate again and finished fermentation in 6 days, the output of active result Antrondin C is 510mg/L, compares with the blank group and has improved 325%.
Embodiment 11:
Identical with the step of the foregoing description four; Different is: in the time of 48 hours, in nutrient solution, adding volume(tric)fraction is 0.5% tensio-active agent Span-80, when cultivating 24 hours in cell cultures; The interpolation volume(tric)fraction is 25% extraction agent; Continue to cultivate again and finished fermentation in 6 days, the output of active result Antrondin C is 350mg/L, compares with the blank group and has improved 190%.
Can know during the fermentation at the foregoing description one to embodiment 11, can effectively improve the cell permeability, make a large amount of being discharged into outside the born of the same parents of active result Antrondin C through preferred surfactants Span-80; Alleviate the negative feedback inhibition effect of intracellular product because active result is insoluble in water, be secreted into the outer active result of born of the same parents be extracted basically enrichment to dodecyl mutually in; So will reconciling the cell permeability, the present invention combines with the original position extraction phase; Set up the best biliquid fermentation system that is coupled, can in same fermentation period, realize the efficient synthetic of active result and extract, reduced production cost; Simplified zymotechnique; Particularly when using 0.5% tensio-active agent Span-80, with 20% extraction agent dodecyl mutually during coupling, the output of active result Antrondin C can reach 510mg/L; Compare with the blank group and to have improved 325%, effect is remarkable.The coupled fermentation system set up of the present invention active result kind of producing horn of plenty more not only contains active result Antrondins compound in addition, also detects active result Antroquinonol class material and coenzyme class material; The fermentation culture cycle shortens with respect to solid-state cultivation greatly in addition; Physiologically active substance kind and content and solid state fermentation obtain quite, solve the single problem of the conventional liquid state fermentation synthetic physiological active products kind of camphor tree sesame, the active result of dodecyl in mutually can come out through alcohol extraction after the fermentation ends; Aftertreatment technology is simple; Yield is high, and the dodecyl after the extraction can reuse after removing residual ethanol through distillation, has certain economic benefits; The personnel of association area can carry out change, replacement and the combination of other various ways according to method provided by the invention, and realizing should technology.
Claims (9)
1. the biliquid phase fermentation method of coupling original position extraction camphor tree sesame active result Antrondin C; After it is characterized in that camphor tree sesame (Antrodia camphorata) bacterial classification is through the liquid seed culture medium enlarged culturing, carry out liquid fermentation and culture, the control rotating speed is 80～180 rev/mins; 22～34 ℃ of temperature; Fermentation culture 8～12 days is added tensio-active agent and extraction agent during the fermentation successively, sets up the biliquid phase fermentation system of regulating the extraction of cell permeability coupling original position; Reduce the intensity of physiological active products negative feedback inhibition, improve camphor tree sesame active result output; Wherein the interpolation time of extraction agent is later than tensio-active agent 12-48 hour.
2. the described method of claim 1 is characterized in that said tensio-active agent is that Span series, Tween are serial, Triton, PEG series or Chinese honey locust.
3. the described method of claim 2, the interpolation time that it is characterized in that tensio-active agent Span-80 is that liquid state fermentation was cultivated the beginning back 24-96 hour, the adding proportion of Span-80 is 0.1%～15%.
4. the described method of claim 3, the interpolation time that it is characterized in that tensio-active agent Span-80 is liquid state fermentation when cultivating 48 hours, adding proportion is 0.5%.
5. the arbitrary described method of claim 1 is characterized in that said extraction agent is long-chain unsaturated fatty acid or long chain alkane class.
6. the described method of claim 5 is characterized in that said extraction agent is a dodecyl.
7. the arbitrary described method of claim 1-6, the interpolation time that it is characterized in that dodecyl is after adding tensio-active agent 12～48 hours, addition is 5%～30%.
8. the arbitrary described method of claim 1-6, the interpolation time that it is characterized in that dodecyl is after adding tensio-active agent 24 hours, addition is 20%.
9. the arbitrary described method of claim 1-6 is characterized in that said liquid fermentation medium composition is: glucose 50～100g/L, analysis for soybean powder 5～15g/L, corn starch 2～6g/L, sal epsom 0.1～1.5g/L, potassium hydrogenphosphate 0.1～1.5g/L.
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| CN105483160A (en) * | 2015-12-31 | 2016-04-13 | 浙江省林业科学研究院 | Antrodia camphorata cultivating composition and preparation method thereof |
| CN108048336A (en) * | 2017-12-30 | 2018-05-18 | 佛山科学技术学院 | A kind of nitrogen source fluid nutrient medium of Antrodia camphorata and its application |
| CN110305918A (en) * | 2019-06-19 | 2019-10-08 | 北京化工大学 | A kind of method of Antrodia camphorata fermentation coupling extraction production 4-Acetylantroquinonol B |
| CN113755356A (en) * | 2021-10-19 | 2021-12-07 | 浙江大学 | Gene engineering bacterium for extracellularly secreting tocotrienol and application thereof |
| CN114790438A (en) * | 2021-12-24 | 2022-07-26 | 上海理工大学 | Method for improving yield and oxidation resistance of antrodia camphorata exopolysaccharide |
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| CN104946699A (en) * | 2014-03-26 | 2015-09-30 | 江南大学 | Double-liquid-phase fermentation method of monascus yellow pigment by coupled in-situ fermentation-extraction |
| CN104946699B (en) * | 2014-03-26 | 2018-02-23 | 江南大学 | A kind of biliquid phase fermentation method for being coupled situ extracting fermentation monascus yellow pigment |
| CN105483160A (en) * | 2015-12-31 | 2016-04-13 | 浙江省林业科学研究院 | Antrodia camphorata cultivating composition and preparation method thereof |
| CN105483160B (en) * | 2015-12-31 | 2019-01-18 | 浙江省林业科学研究院 | A kind of Antrodia camphorata culture composition and preparation method thereof |
| CN108048336A (en) * | 2017-12-30 | 2018-05-18 | 佛山科学技术学院 | A kind of nitrogen source fluid nutrient medium of Antrodia camphorata and its application |
| CN110305918A (en) * | 2019-06-19 | 2019-10-08 | 北京化工大学 | A kind of method of Antrodia camphorata fermentation coupling extraction production 4-Acetylantroquinonol B |
| CN113755356A (en) * | 2021-10-19 | 2021-12-07 | 浙江大学 | Gene engineering bacterium for extracellularly secreting tocotrienol and application thereof |
| CN114790438A (en) * | 2021-12-24 | 2022-07-26 | 上海理工大学 | Method for improving yield and oxidation resistance of antrodia camphorata exopolysaccharide |
| CN114790438B (en) * | 2021-12-24 | 2024-02-27 | 上海理工大学 | Method for improving yield and oxidation resistance of antrodia camphorata extracellular polysaccharide |
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